应用RNA干扰下调多药耐药蛋白4表达对结直肠癌细胞照射敏感性的影响

目的探讨多药耐药蛋白4（MRP4）表达对结直肠癌细胞照射敏感性的影响。方法应用慢病毒感染RNA干扰技术（RNAi）稳定下调人结直肠癌细胞株HCT116中MRP4的表达。将HCT116细胞分为未感染任何病毒的细胞株（CON）、加阴性对照病毒感染的细胞株（NC）和加RNAi靶点病毒感染的细胞株（KD）3组,应用实时定量RT-PCR和Western blot分别从RNA和蛋白水平检测MRP4表达变化,以验证RNAi的有效性。4Gy剂量照射后24h,流式细胞术检测细胞凋亡,MTT检测细胞增殖,比较RNAi后HCT116细胞株照射敏感性的差异。结果成功构建并转染慢病毒质粒,获得稳定沉默MRP4表达的HCT116-KD细胞株,HCT116-KD细胞株MRP4mRNA和蛋白表达水平较对照均明显下调（P〈0．05）。照射后24h,KD细胞株凋亡率为（71．7±0．8）％,明显高于CON组（56．1±0．9）％]和NC组（59．8±0．8）％]（P〈0．05）。照射后48h和72h,KD细胞增殖能力较对照组明显下降（火0．05）。结论MRP4在结直肠癌细胞中的表达水平与照射耐受显著相关,应用慢病毒感染RNA干扰下调MRP4表达可以增强结直肠癌细胞照射敏感性。MRP4有可能成为预测放疗敏感性的分子标志物。

Down regulation of multidrug resistance-associated protein 4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro

Objective To investigate the effect of multidrug resistance-associated protein 4 （MRP4） expression on the radiosensitivity of coloreetal carcinoma cell lines in vitro. Methods The vector of shRNA for RNA interference was constructed and then transfected into HCTll6 cell line to steadily down-regulate the expression of MRP4. HCTll6 cells were divided into 3 groups including the CON group （non-transfected）, NC group （negative control virus was added）, and KD group （RNAi target was added for transfection）. To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow eytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTI＂ assay at different time points. Results ShRNA plasmid was successfully constructed. Transfeetion of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression （HCT116-KD）. MRP4 mRNA and protein expression were significantly down-regulated following RNA interference （P〈0.05）. Twenty-four hours after radiation, the apoptosis rate of KD cell line was （71.7±0.8）%, significantly higher than that in the CON group （56.1±0.9）%]and NC group （59.8±0.8）% ] （P〈0.05）. Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups （P〈0.05）. Conclusions Expression of MRP4 is closely related to radiotolerance of colorectal carcinoma. Downregulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.